Blood Draw Tube Colors and Order

The tube order may not seem like a big deal and may seem unnecessary to some, but it is very important to pay attention too. It also matters as to what type of needle is being used for the draw. If a butterfly needle is being used it is important to have a spit tube because with a butterfly there is air within the hose that connects the needle to the vacutainer. Its important to get this air out before filling any tubes used for patient testing. If a standard needle is being used, you typically don’t need the spit tube, but its good practice. The order still remains the same for each.

Light Blue: The typical tube for routine coagulation studies. The additive is sodium citrate (3.2% or 3.8%). Citrate is a anticoagulant which binds to calcium within the blood so the blood can’t clot. Calcium plays an important role in primary and secondary homeostasis. See my post on DIC for that information, in short it is used in the coagulation cascade. An important aspect of coagulation studies is that the light blue tube must, must be filled completely. There is a ratio of sodium citrate to whole blood and that must remain constant. The tube must be rejected if it is not filled completely.

Green or Mint Tubes: These tubes are used for chemistry studies. Often referred to as PST or plasma separator tubes. The additive in these tubes are sodium heparin, lithium heparin or ammonia heparin. The heparin, being an anticoagulant activates antithrombin, which blocks the coagulation cascade and produces a whole blood with plasma sample instead of a clotted blood and serum sample. When these tubes are centrifuged, the gel barrier moves upwards creating a barrier that separates the plasma from the red cells allowing the plasma to be aspirated directly for testing.

Gray Tube: The gray tube tops are typically used for glucose testing, ethanol levels or lactate level testing. The additive is potassium oxalate and sodium fluoride. Potassium oxalate is an anticoagulant which prevents clotting and the sodium fluoride is an anti-glycolytic which prevent the cells from using the glucose in the sample.

Lavender/Pink Tube: The lavender tube is typically used for hematological testing or for Hemoglobin A1C testing. The pink tube is used primarily for blood bank testing such as type and screen and cross-matching. The additives in the lavender and pink tubes are EDTA K2 or EDTA K3. The EDTA binds to calcium which blocks the coagulation cascade in the same way that citrate in the light blue tube does. Red cells, leukocytes, and platelets are in EDTA anticoagulated blood for 24 hours. Blood smears should be done within 3 hours of receiving the sample.

SST/Mustard Tube: Serum separator and clot activator that will separate the blood from the serum upon centrifugation. This tube is usually used to test for aldosterone, B12, ferritin and folate levels.

There are not all the different tubes that are used, but these are the most common tubes that I listed and the ones that a laboratory professional will most likely come across. Its important to understand the additive in each one to make sure that they are appropriate for the testing that needs to be done on the patient sample itself within the tube.

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-Caleb

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Transfusion Reactions

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The blood bank of any laboratory deals with a huge responsibility. They play a role in the initial compatibility testing of blood donor products and the recipient or patient serum. The patients serum contains naturally occurring antibodies or in certain circumstances where the patient has had a previous transfusion, the serum can contain alloantibodies that have been synthesized from previous donor blood products. Research has progressed suggesting that whole blood donor products are not as effective at replacing volume as individual components are. When a donor comes in and donates a pint of blood there are techniques that are used to separate the plasma from the blood products. Platelets are collected via an apheresis machine. When the plasma is separated out it must be frozen at >-20 degrees C within 8 hours of collection. When a patient needs plasma, it takes about 18-20 minutes to thaw and release. Fresh frozen plasma (FFP) has an expiration off 12 months.  Red blood cells are usually stored in a refrigerator at 1-6 degrees C. RBC products have an expiration of 42 days once collected. They can be frozen for 10 years if needed.

For transfusion purposes compatibility needs to be done correctly and cautiously. Platelets do not need to be ABO or Rh compatible, but if ample supply is available, its best to ABO match donors with the patient. Red blood cells absolutely need to be ABO and Rh compatible. If a compatible unit is not available then the hospital should use an O negative unit. O negative units are used as the universal donor. Plasma should be ABO compatible. Contrary to RBCs units, an AB plasma donor is considered the universal donor where in RBC products an O negative donor is the universal donor as mentioned previously. Plasma products contain the donors antibodies. When the donor is AB, they do not have anti-A, or anti-B. It is because of this principle that AB plasma is considered as the universal donor.

Even when every precaution is taken to ensure proper testing took place and compatibility testing was as objectively accurate as possible transfusion reactions can still take place. There is no way to 100% prevent them. Acute hemolytic reactions are typically the most severe and occur when ABO-incompatible blood is given. With acute hemolytic reactions fever and chills develop quickly, back and flank/pain (Renal failure) can occur with hemoglobinuria/hemoglobinemia. Bleeding and DIC can commonly be seen. Treatment is to stop transfusion immediately and volume replacement. Diuretics may be given, most commonly furosemide. Febrile non-hemolytic reactions are typically caused by transfusion of leukocytes that attack the recipient. A fever that is characterized as greater than 1 degree Celsius increase. The infusion of the leukocytes cause cytokine release such as IL-6, and TNF. Transfusion of HLA antibodies can occur as well. Antipyretics can be given to resolve. It is also recommended to infuse leukocyte reduced units in the future.

Bacterial contamination can occur which can cause sepsis. Typically there will be a rapid high fever with symptoms of rigor, shock and gastro symptoms. Bacterial contamination usually is able to be cultured from the donor bag along with the collection site. Antibiotics should be administered with support as necessary. A way to get around this is to leukoreduce donor units.

Transfusion-related Acute Lung Injury (TRALI) is an acute lung injury <6 hours after transfusion that presents with hypoxemia and lung infiltrates. The anti-HLA antibodies activate the PMNs in the lung endothelial which causes physiological stress. TRALI is 20% fatal, but treatment should be aggressive supportive care with fluids.

Acute afebrile reactions include allergic, anaphylactic, and transfusion associated circulatory overload (TACO) reactions. A typical urticarial or allergic reaction presents with localized hives/redness which is caused by a IgE hypersensitivity. Typical treatment includes antihistamines. Anaphylactic reactions are caused by anti-IgA antibodies in the recipient. Usually signifying that the recipient is also IgA deficient. Presents with hypotension, GI symptoms and fever with anti-IgA. Treatment is immediate epinephrine or transfusion with washed RBCs or platelets. TACO usually occurs with a history of cardiopulmonary disease with too rapid of blood infusion. High risk groups include the elderly and adolescents. TACO presents with dyspnea, and hypoxia during and after transfusion. Elevated BNP, JVD and BP. Treatment is to slow the rate of infusion and diuretics.

Delayed febrile reactions typically present greater than one week post transfusion. There is a positive DAT along with hyperbilirubinemia and evidence of a new alloantibody. Delayed febrile reactions are caused by a anamnestic response to re-exposure to red cell antigens. Treatment is support therapy. Graft-vs-host-disease (GVHD) is caused by cellular immune response by transfused T-lymphocytes versus the host or recipient. Presentation includes fever, diarrhea, skin rash. Treatment includes immunosuppressive therapy with supportive care. GVHD can be 90% fatal.

Delayed afebrile reactions include post transfusion purpura and iron overload. PTP is caused by a recipient antibody versus the absent platelet antigen (HPA-1a). There is a decrease in platelets, and increased bleeding. Treatment includes IVIG and plasma exchange. Its important to avoid platelet transfusion. Iron overload typically occurs when >100 units have been transfused. Liver, pancreas, and cardiac dysfunction occurs. Iron chelation is standard treatment.

All reactions are serious and should be treated as such. Its important to check for clerical error in pre-transfusion compatibility testing as that is the number one cause of transfusion related reactions.

ABO Discrepancies

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ABO discrepancies can happen for a variety of reasons and it is important to learn to recognize a discrepancy and what to do to correct it.

Clerical and technical errors are by far the most common ABO discrepancy and are usually fairly easy to correct. It is important to record the results of each reaction as you read each tube individually, don’t wait to record the results at the end as this increases the chance of recording an incorrect results. Another common error is writing down the results for the wrong patient, it is important that one patient is run at a time as this gives the Technologist the time to devote to each individual patient.

Failure to add serum or reagent can lead to technical errors and failure of a reaction to occur when one is expected. One of the golden rules of blood bank is to add the reagent antisera and serum BEFORE adding the patients or reagent red cells. Contaminated reagents can lead to erroneous results such as false positives or false negatives. It is important to centrifuge properly as with under centrifugation can alter antibody binding. Over centrifugation can lead to false positives while reading the reaction while there is still a button on the bottom of the tube. ABO antibodies are IgM which react between 4-20 degrees Celsius. Warming the reaction can cause a false negative.

Weak or missing antibodies can occur for many reasons. Newborns don’t develop antibodies until at least 6 months of age. Elderly individuals lose the ability to maintain antibody levels so they may appear weak or missing upon serum testing. Certain patient conditions such as immune deficiencies, chemotherapy, radiation therapy, and bone marrow transplantation explain the discrepancy.

Resolution is through adding two drops of serum in the event that it wasn’t added the first time. Then centrifuge. If still negative then incubate at 4-18 degrees Celsius for 15-30 minutes.

Rouleaux formation can cause unexpected agglutination in all serum tests. Rouleaux is a phenomenon as a result of multiple pathologies such as multiple myeloma, macroglobulinemia, and liver disease. Rouleaux will appear as agglutination macroscopically, but microscopically will appear as stacks of coins. Resolution is through saline replacement.

Weak or missing antigens can be due to the patient having a weak subgroup of A or B, acute leukemia, massive transfusions (Group O), or history of bone marrow transplant. Care should be taken to receive recent transfusion history and any other clinical history. Read the forward typing microscopically to confirm any reactions. Use Anti-A,B and incubate at 4-22 degrees Celsius for at least 15 minutes. Monoclonal antisera can be used that reacts with weak subgroups of A and B.

The acquired B phenomenon can be see in patients who have problems with the colon or infections with gram-negative rods such as E. coli. Bacterial enzymes modify the A antigen to a B antigen and can cause forward typing AB and reverse typing as A. Resolution is to check history for colon infections. Setting up an auto-control can distinguish between a true B and acquired B. The patients own Anti-B will not cause agglutination with the AB cells. Putting the anti-B reagent in a pH 6 solution will acidify it. After re-testing the acquired B antigens will not react with the antisera. Normal B antigens will.

Polyagglutinable cells such as Whartons Jelly, which is found in cord blood will show discrepancies in the forward typing. Whartons Jelly coats the newborns blood so the child may appear type AB. It is important to re-type the newborn after washing 4-5 times to remove the Whartons Jelly.

-Caleb